1. Field of the Invention
This invention is directed to methods for the enzymatic synthesis of alpha-sialylated oligosaccharide glycosides. Specifically, in the methods of this invention, sialyltransferase is employed to transfer an analogue of sialic acid, employed as its CMP-nucleotide, to an oligosaccharide glycoside. The analogue of sialic acid and the oligosaccharide glycoside employed in this method are selected to be compatible with the sialyltransferase employed.
2. References
The following references are cited in this application as superscript numbers at the relevant portion of the application:
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3. State of the Art
Carbohydrates and/or oligosaccharides are present on a variety of natural and pathological glycoconjugates.sup.1. Of particular interest are carbohydrates and oligosaccharides containing sialic acid residues particularly at the nonreducing sugar terminus.sup.31. Such sialic acid terminated carbohydrates and oligosaccharides are present in a number of products which have been implicated in a wide range of biological phenomena based, in part, on the concept of recognition signals carried by the carbohydrate structures and by their binding to specific ligands.
Specifically, such sialic acid terminated carbohydrates and oligosaccharides are believed to be receptors for the binding of toxins.sup.4, pathogenic agents such as viruses.sup.5, and are believed to be recognition sites for a variety of lectins, particularly those involved in cellular adhesion.sup.6,7, etc.
Similarly, certain oligosaccharides including sialic acid terminated oligosaccharides have been identified as capable of suppressing a cell-mediated immune response to an antigen. The ability of such oligosaccharides to suppress a cell mediated immune response to an antigen is described by Ippolito et al..sup.3 which reference is incorporated herein by reference in its entirety.
Additionally, the presence of certain sialyl terminated oligosaccharides in tumor-related antigens is documented in the art.sup.1 and, in general, the structures of the oligosaccharides present on such antigens have been modified in some way from normal oligosaccharides so as to lead to the expression of tumor related antigens.sup.2. The prospect of passive immunotherapy with monoclonal antibodies directed against some sialylated tumor-associated antigens, such as the gangliosides GD.sub.2, GD.sub.3 and GM.sub.2, in patients with melanoma is being investigated.sup.8,9. However, most tumor-associated antigens are unable to lead to the production of tumor specific antibodies which would either inhibit or prevent the growth of such tumors. Without being limited to any theory, it is believed that this is due to the absence of real tumor specific antigens and that the structure of such antigens cross-react with that of similar structures expressed in a restricted number of normal tissue. In addition, carbohydrate antigens are generally not believed to lead to a T-cell mediated immune response that is expected to play a role in active immunity.sup.10. However, some recent studies indicate that, in some cases, tumor-associated carbohydrate antigens may stimulate anticancer T-cell immunity.sup.11,12 or the production of cytotoxic antibodies.sup.13.
In view of the general inability of carbohydrate tumor-related antigens to produce cytotoxic tumor specific antibodies, it has been proposed to chemically modify naturally occurring weak antigens so as to improve their antigenicity.sup.14. In this regard, methods for chemical modification of specific groups on carbohydrate tumor-related antigens have been reported and, because of the importance of the sialic acid group in sialylated oligosaccharide antigens, much of the focus of chemical modifications to non-or weakly-immunogenic or naturally occurring antigens has been to derivatize the sialic acid residue in the expectation that such modification could result in improved immunogenicity.
Specifically, the art has recognized that some structural modifications present on naturally occurring sialic acids render the corresponding oligosaccharides immunogenic in selected hosts. For example, E. coli K1 polysaccharide [an alpha(2-8) linked polymer of N-acetylneuraminic acid] is a poor immunogen but antigenic variations resulting from a partial and random O-acetylation of the sialic acid gave polysaccharides with increased immunogenic properties..sup.15 Chemical modifications of the sialic acid moiety of rabbit transferrin provides a modified antigen that produced cross-reacting auto-antibodies in rabbits.sup.16. Likewise, the creation of a more immunogenic epitope by chemical modification of the sialic acid group of B meningococcal polysaccharide has been achieved by the replacement of the N-acetyl with an N-propionyl group. The resulting artificial antigens produced high levels of cross-reacting antibodies as well as imparting a boosting effect.sup.17. Other literature references disclose immunogenic properties for the ganglioside GM.sub.1.sup.18,19 and GM.sub.3.sup.82 obtained by chemical modification of the sialic acid group.sup.18,19.
Recent work with artificial antigens indicates that while chemically modified sialosides (melanoma associated glycolipid antigens) are antigenic in humans, the antibodies generated by these modified sialosides do not cross-react with the natural substance..sup.81 On the other hand, when injected into mice, chemically modified sialylated antigens produce antibodies which do cross-react with the natural substance. Accordingly, cross-reacting monoclonal or polyclonal antibodies generated in mice would serve as a basis for either a diagnostic assay for determining the presence and/or amount of the natural substance in a human host or as a basis for antibody therapy for a disease condition in which the natural substance is attack by the antibodies which can optionally be coupled to a therapeutic agent.
It is also contemplated that the chemical modification of the sialic acid group of sialylated oligosaccharide glycosides could result in oligosaccharide glycosides having improved activity in the suppression of cell-mediated immune responses to an antigen.
However, chemical modification of the sialic acid group of such antigens or of such oligosaccharides is not feasible on a practical level by virtue of the fact that the chemical reactions employed must be specific for the intended modification so that the antigen or oligosaccharide is not altered in some unintended manner. As is apparent, such limitations make impractical the extent and type of chemical modifications which can be conducted on oligosaccharides or antigens.
Alternatively, it is also possible to initially modify the sialic acid which is then coupled to an oligosaccharide glycoside by chemical synthesis so as to provide an alphasialylated oligosaccharide containing an analogue of sialic acid. In this approach, a modified sialic acid residue is chemically generated and then chemically added to a specific site of an oligosaccharide. The modified oligosaccharide could either be used in methods for suppression of cell-mediated immune responses; or, if an appropriate aglycon group is employed, the modified oligosaccharide could be linked to a carrier to generate an artificial conjugate, including artificial antigens.
However, this approach is complicated by the fact that such a chemical synthesis invariably involves multi-step procedures which, at best, generally leads to overall moderate yields in the final product. Additionally, the inherent difficulties in chemically making stereoselectively an alpha-sialoside linkage dictate that the complete chemical synthesis of alpha-sialylated oligosaccharides would be a lengthy and a particularly difficult process for modified sialic acids.
In view of the above, it would be particularly advantageous to develop methods for the facile preparation of alpha-sialylated oligosaccharides containing an analogue of sialic acid. The present invention accomplishes this by using sialyltransferases to effect efficient coupling of an analogue of sialic acid activated as its CMP-nucleotide derivative (an artificial donor as defined below) to a saccharide glycoside or an oligosaccharide glycoside (an artificial acceptor as defined below).
While admittedly sialyltransferase enzymes are known to transfer N-acetylneuraminic acid (natural donor), activated as its cytidine monophosphate (CMP) derivative, to the terminal oligosaccharide structures of glycolipids and glycoproteins (natural acceptors), the use of such transferases in the transfer of an analogue of N-acetylneuraminic acid (artificial donor) to the non-reducing sugar terminus of an oligosaccharide glycoside (artificial acceptor) and other artificial acceptors has not heretofore been disclosed. At best, the art discloses that sialyltransferases can accept modification in either the donor or the acceptor and still result in the transfer but the art fails to suggest that sialyltransferases can accept modification in both the donor and the acceptor and still efficiently transfer analogues of sialic acid. The finding that sialyltransferases possess sufficient recognition flexibility to transfer an artificial donor to an artificial acceptor is particularly surprising in view of the unpredictability of catalysis including enzyme catalysis.